Page 184 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

P1: GPB Final Pages
Encyclopedia of Physical Science and Technology EN013D-617 July 27, 2001 11:42

224 Protein Synthesis

IleRS:Val-AMP + tRNA Ile → IleRS + Val tween the halves. As described earlier, this polypeptide
makes up the editing domain present in some enzymes.
Ile
+ AMP + tRNA (Pretransfer)
Comparison of Class I enzymes identified a signature se-
IleRS:Val-tRNA Ile → IleRS + Val quence within the active site; this stretch of 11 amino acids
is located in the first half of the nucleotide binding fold and
Ile
+ tRNA (Post-transfer).
ends in HIGH (the one-letter code for His–Ile–Gly–His).
Both activities are dependent on the presence of cog- The second half of the fold contains another conserved se-
Ile
nate tRNA , particularly the D-arm, as determined by quence, KMSKS, which also makes critical contributions
studies that investigated the ability of mutants of tRNA Ile to the enzyme active site.
to trigger editing. Furthermore, the editing site on IleRS Class II AARSs were first distinguished by their lack of
has been located within a peptide (connective polypep- the Class I signature sequence, but this subfamily also has
tide 1, CP1) inserted between the two halves of the Ross- distinct features of its own. They are mostly α 2 dimers, al-
mann nucleotide binding fold that makes up the catalytic though some are α 2 β 2 tetramers. The enzymes belonging
domain of Class I AARSs (see following). Valyl–tRNA to Class II have active sites composed of a seven-stranded
synthetase (ValRS) and leucyl–tRNA synthetase (LeuRS) antiparallel β-sheet with three α-helices. The characteris-
have sequences in CP1 similar to IleRS; it is interesting tic Class II sequence motifs 1, 2, and 3 exhibit only min-
to note that these enzymes all recognize amino acids with imal sequence conservation, but contribute a helix-loop-
aliphatic side chains. Isolated CP1 domains from IleRS strand, strand-loop-strand, and strand-helix to the active
and ValRS have been shown to execute post-transfer site, respectively.
editing. A mechanistic difference between the enzymes of
The location of the editing site in IleRS has been Classes I and II is the site of initial amino acid attach-
visualized by X-ray crystallography and is approximately ment to the tRNA. Class I enzymes aminoacylate the
˚

25 A from the “synthetic” active site. Apparently both 2 -hydroxyl of the terminal adenosine’s ribose and

the aminoacyl adenylate and the aminoacylated tRNA most Class II enzymes use the 3 -hydroxyl. The amino
must be translocated from the synthetic active site to the acid subsequently migrates between the two positions.
editing active site for an accuracy check before being This early functional observation was rationalized once
released. Translocation of the misaminoacylated tRNA high-resolution enzyme–tRNA cocrystal structures were
to the editing site, and subsequent proofreading, has been determined. Class I enzymes approach the tRNA acceptor
proposed to be triggered by a conformational change in stem from the minor-groove side of the RNA helix and

the tRNA. How the isolated aminoacyl adenylate is are nearer the ribose 2 -hydroxyl, while Class II enzymes
translocated remains unknown. The overall mechanism approach from the major-groove side of the tRNA, near

for accurate aminoacylation by IleRS has been termed a the 3 -hydroxyl.
“double-sieve”—amino acids that don’t fit into the active
site binding pocket are excluded based on size, while
F. Mechanistic Clues from Structural Studies
valine and smaller amino acids are actively hydrolyzed at
the second (editing) site when they are misactivated or Enzyme–tRNA cocrystal structures have also provided
Ile
subsequently attached to tRNA . clues to the catalytic mechanisms used by AARSs. In the
case of the Class I glutaminyl-tRNA synthetase (GlnRS)
complexed with its cognate tRNA Gln , the structure of
E. Class Organization of AARSs
the complex identified variations in the tRNA architecture
Detailed structural information from X-ray crystal- compared with uncomplexed tRNA. GlnRS makes essen-
lographic studies is available for nearly all of the 20 tial contacts with nucleotides in the tRNA Gln anticodon,
AARSs. As more and more sequences and structures were and in the cocrystal structure these nucleotides were in-
determined the synthetases were seen to be partitioned deed splayed out from their normal positions to interact
into two classes of 10 enzymes each, based on similarities closely with amino acids of the enzyme. Furthermore, the
in their catalytic cores. acceptor end of the tRNA showed significant distortion
Enzymes belonging to Class I function as monomers or compared to the expected helical structure. The first base
homodimers (α 2 ). Their active sites contain a Rossmann pair of the acceptor helix was broken, and the 3 -end of the

nucleotide binding fold, also observed in dehydrogenases tRNA bent back in a hairpin conformation toward the rest
and other nucleotide-binding proteins. This fold consists of the helix (Fig. 3). Such a folded-back orientation of the

of an alternating pattern of β-strands and α-helices. The acceptor end of tRNA Gln is necessary for the 3 -terminus
nucleotide binding fold is split into two halves, with a to reach the glutaminyl adenylate in the enzyme active site.
variable-length polypeptide insertion known as CP1 be- Furthermore, amino acid residues in the enzyme’s active